Photobleachable luminescent layers for calibration and standardization in optical microscopy

ABSTRACT

The invention pertains to a calibration layer comprising an optically transparent polymer containing an amount of photobleachable luminscent material present in such a way that the final polymer film contains less than 10 wt. % of luminophore and has an optical attenuation of less than 0.3 absorption units in the wavelength region of interest. The invention further is concerned with a method of calibration of an optical image device, preferably an optical or Raman microscope, by using the decrease in luminescence as the result of photobleaching between two consecutive images for calibration.

BACKGROUND OF THE INVENTION

The invention pertains to the preparation and use of thin,photobleachable luminescent layers for calibration and standardizationof optical imaging devices, such as optical or Raman microscopy. For thequantitative application of optical and Raman microscopy, it isessential that the intensities in the images acquired with thesemicroscopic techniques are determined only by the spatial distributionof the concentration, absorption, and emission characteristics of theluminophores in the specimen under investigation. If this is notpossible, the image intensities should at least be proportional to theseparameters. Generally, however, image intensity variations are not onlydetermined by the specimen, but also by spatial non-uniformities of theoptical system of the microscope across the field of view, so that onlyqualitative investigations can be performed. In order to realize theimages required for quantitative microscopy, the microscope must becalibrated and standardized. The thus obtained images allow thecomparison of different samples obtained on the same microscope, also ata different point in time, or the comparison of images obtained ondifferent microscopes, provided that the different microscopes have beencalibrated in the same way.

In earlier work, calibration and standardization of an opticalmicroscope was attempted by an approach using images of a uniformluminescent layer (K. R. Castleman, Digital Image Processing.Prentice-Hall, Englewood Cliffs, N.J., 1979, and Z. Jericevic, B. Wiese,J. Brayan & L. C. Smith, “Validation of an Image System,” inLuminescence Microscopy of Living Cells in Culture, Part B, QuantitativeLuminescence Microscopy-Imaging and Spectroscopy, edited by D. LansingTaylor and Y. Wang, Academic Press, San Diego, Calif., 1989). Such anapproach has three disadvantages. Firstly, in the case of an image of aluminescent layer, the product of the illumination and detectionefficiency distributions is measured, and no information on the separatedistributions is available. Secondly, completely uniform luminescentlayers are difficult to obtain. Thirdly, the results of calibration andstandardization based on this approach are affected by luminescencephotobleaching of the layer. For general calibration and standardizationin optical microscopy, it would be preferable to have an approach whichdoes not suffer from these disadvantages. Jericevic et al. (Z.Jericevic, B. Wiese, J. Brayan & L. C. Smith, “Validation of an ImageSystem,” in Fluorescence Microscopy of Living Cells in Culture, Part B,Quantitative Fluorescence Microscopy-Imaging and Spectroscopy, edited byD. Lansing Taylor and Y. Wang, Academic Press, San Diego, Calif., 1989)attempted to do away with the first disadvantage by using luminescencephotobleaching techniques for the determination of only the illuminationdistribution. In his method, at least 20 images of a uniform,photobleaching luminescent layer were required. They showed that bynumerically fitting the luminescence intensity decrease in each pixel ofthe first image with an exponential function, it was possible todetermine only the excitation intensity distribution in the field ofview of the used microscope (Z. Jericevic, D. M. Benson, J. Bryan, & L.C. Smith, “Rigorous Convergence Algorithm for Fitting a MonoexponentialFunction with a Background Term Using the Least-Squares Method,” Anal.Chem., 59 (1987), 658-662). There are several drawbacks to this methodand experimental approach. Firstly, a luminescent layer has to beprepared by spreading an FITC-IgG mixture on a microscope slide. Withsuch a procedure, it is very difficult to obtain a uniform luminescentlayer. Secondly, the method provides only the illumination distribution;no information about the detection distribution is obtained. Thirdly,the determination of the illumination distribution is based onnumerically fitting routines, which renders the method relatively slow.

BRIEF SUMMARY OF THE INVENTION

This invention describes the preparation and use of thin,photobleachable luminescent films for the calibration andstandardization of an optical or Raman microscope in the wavelengthregion of 250 nm-1700 nm, preferably 250 nm-1100 nm, and more preferably350 nm-900 nm.

DETAILED DESCRIPTION OF THE INVENTION

This is achieved according to the invention by the preparation of aphotobleachable luminescent calibration layer and its subsequent use forthe determination of excitation intensity and detection efficiencydistributions in the field of view of the used microscope. The termphotobleaching comprises all processes which result in the reduction ofthe intensity of luminescence light generated at the wavelength ofexcitation. Excitation may be done by laser or by a focused light sourcein the wavelength ranges defined above. Examples of such processes arephoto-oxidation, photo-reduction, photo-isomerization or photo-additionreactions, or light-induced electron transfer processes.

It is sufficient for the effectiveness of the invention that theprepared calibration layer is approximately uniform, luminescent, andphotobleachable, preferably approximately uniform, luminescent, andmono-exponentially photobleachable in a certain regime. The calibrationlayer should satisfy the following requirements.

(i) The luminescence intensity of luminophore in the calibration layershould be proportional to the excitation intensity, the luminophoreconcentration, and the illumination duration.

(ii) The rate of photobleaching of the luminophore in the calibrationlayer should be proportional to the illumination intensity andindependent of the luminophore concentration.

(iii) The luminescence quantum yield, the absorption cross-section, andthe bleach factor—defined as the ratio of the rate of photobleaching tothe excitation intensity—of the luminophore in the calibration layershould be independent of the micro-environment within the layer.

The first two requirements already suffice for qualitative calibrationof the measurement. The third requirement in combination with the firsttwo allows absolute measurement of an image in optical or Ramanmicroscopy.

The calibration layer is applied on an optically flat and transparentsubstrate by spin-coating, dip-coating or rod-coating (doctor blading)of a, preferably, 1-30 wt % solution of an optically transparent polymercontaining an amount of photobleachable luminescent material present insuch a way that the final polymer film contains less than 10 wt % ofluminophore and has an optical attenuation of less than 0.3 absorptionunits in the wavelength region of interest, or of a solution of asidechain polymer with an amount of photobleachable luminescent groupscovalently attached to it, in such a way that the relative molar contentof the sidechains is lower than 10% and the optical attenuation of thecalibration layer is less than 0.3 absorption units in the wavelengthregion of interest. The useful concentration region is determined by thenecessity to prevent intermolecular interactions (energy transfer) andinner filter and concentration quenching effects, which may lead todeviations tom simple mono-exponential decay. Optical attenuation morethan 0.3 absorption units is possible, but mathematical corrections arerequired. Such attenuation is therefore less preferred. Suitablepolymeric materials, which are transparent across the wavelength regionof interest, are polyacrylates, polymethacrylates, polycarbonates,polyolefins, polyethers, polyurethanes, polyetherketones, polyesters,polystyrenes, polysiloxanes, and the like, or copolymers thereof.Suitable polymeric sidechain materials are based on the same opticallytransparent building blocks as applied in the transparent polymer typesmentioned above and a suitable luminescent and photobleachable moleculewhich is equipped with a functional group so that it either may beattached to said polymer or may react with other functional monomers toform a luminescent sidechain-main chain polymer. Alternatively, thinfilms may be prepared by making use of sol-gel glass formationapproaches.

The luminescent material used should be photobleachable, which meansthat the intensity of luminescence should be reduced by illumination inthe microscope at the applied excitation wavelength. A number oflight-induced processes may result in such photobleaching; some examplesare photo-oxidation, photo-reduction, photo-isomerization orphoto-addition reactions, or light-induced electron transfer processes.All luminescent photochromic materials may be used. The photobleachableluminescent material may undergo such change either reversibly orirreversibly.

In view of the excellent homogeneity of the luminescent layers obtainedaccording to the above-mentioned procedure, even the direct luminescenceintensity can be used for calibration.

With the prepared calibration layer, absolute excitation intensity anddetection efficiency distributions in the field of view of the usedmicroscope can be determined from images, before and after partialphotobleaching, of the calibration layer and the luminescence quantumyield, the absorption cross-section, and the bleach factor of theluminophore in the calibration layer, as follows.

When the luminescence intensity of the calibration layer is proportionalto the excitation intensity, the luminophore concentration, and theillumination duration, when its photobleaching is mono-exponential, andwhen its rate of photobleaching is proportional to the excitationintensity and independent of the luminophore concentration, an image ofthe layer acquired before any photobleaching has taken place, referredto below as the “first image,” can be written as the product of theimage exposure time, the luminescence quantum yield, the absorptioncross-section, the bleach factor, and the concentration distribution ofthe luminophore in the calibration layer, and the excitation intensityand detection efficiency distributions of the used microscope. Thedetection efficiency includes all elements of the detection pathwayimportant for the conversion of the intensity to be detected up to theintensity value of a pixel in the final image, such as the finitecollection solid angle of the objective lens, the reflectivity andtransmittance of the optical elements in the detection pathway, and thequantum efficiency of the detector. An image, referred to below as the“second image,” acquired after the calibration layer has been bleachedduring a certain time interval, can be written as the product of thefirst image and an exponential function which is determined by thebleach factor, the excitation intensity, and the bleach time interval.

Based on these two images the relative excitation intensitydistribution—or a distribution proportional to the excitation intensitydistribution—in the field of view of the used microscope can bedetermined by calculating the logarithm of the ratio between the firstand second images of the calibration layer. The absolute excitationintensity distribution can be determined by calculating the ratio of therelative excitation intensity distribution and the bleach factor of theluminophore in the calibration layer to the bleach time interval. It isimportant to point out that for the determination of this—relative orabsolute—excitation intensity distribution, it is not required that thecalibration layer is uniform.

Once the relative excitation intensity distribution has been determined,the relative detection efficiency distribution—or a distributionproportional to the detection efficiency distribution—can be determinedas follows. Firstly, a distribution proportional to the product of thedetection efficiency and luminophore concentration distributions,referred to below as the “product distribution,” is determined bycalculating the ratio of the first image to the relative excitationintensity distribution. Secondly, a number of product distributions aredetermined from the same number of image pairs, first and second images,with each image pair acquired from a different part of the calibrationlayer. By averaging these different product distributions, thecontribution of possible non-uniformities of the luminophoreconcentration distribution can be eliminated, and a distributionproportional to only the detection efficiency distribution, i.e., therelative detection efficiency distribution, is obtained. The number ofproduct distributions required for averaging depends on the uniformityof the calibration layer: for uniform layers, no averaging is required,but the less uniform the layer is, the larger the number of differentproduct distributions should be. For many applications the determinationof the relative distributions is already sufficient.

When the direct luminescence intensity is used, the excitation intensitydistribution and the detection efficiency distribution cannot bedetermined separately. For many applications, e.g., shadow correctionprocedures, it is sufficient to use the product of the intensitydistributions for calibration purposes.

The absolute detection efficiency distribution can be determined bycalculating the product of the relative detection efficiencydistribution, the bleach factor of the luminophore in the calibrationlayer, and the bleach time interval, and dividing the result by theimage exposure time and the luminescence quantum yield, the absorptioncross-section, and the mean luminophore concentration of the luminophorein the calibration layer. The parameters which have to be known forabsolute determination of the excitation intensity and detectionefficiency distributions are the absorption cross-section, theluminescence quantum yield, and the bleach factor of the calibrationlayer. All three parameters can be determined independent of themicroscope used.

The absorption cross-section of the calibration layer at a certainwavelength can be determined by measuring the optical attenuation at thesame wavelength and combining this information with the thickness of thelayer and its luminophore concentration.

The luminescence quantum yield of the calibration layer can bedetermined through comparison of the luminescence of the calibrationlayer with the luminescence of a reference layer of which theluminescence quantum yield is known.

The bleach factor of the calibration layer can be determined bymeasuring the relative decrease of the luminescence intensity afterillumination with a known excitation dose, i.e., energy per unit area.

With the excitation intensity and detection efficiency distributionsknown, a number of calibration and standardization steps in opticalmicroscopy are available.

(i) The method can be employed to compare the excitation intensity anddetection efficiency distributions of different microscopes, or of thesame microscope at different points in time. Differences between theoverall performance of microscopes can be attributed to the excitationpathway, the detection pathway or both. Such information can be used toselectively optimize the pathway that limits the performance. Anotherpossibility is to adjust the illumination and detection parameters ofdifferent microscopes in such a way that equal—or at leastcomparable—excitation and detection conditions result This facilitatesthe comparison of measurements in which one (type of specimen is studiedeither with different microscopes or with the same microscope atdifferent times.

(ii) The excitation intensity distribution is important in theinterpretation of the intensity variations in images obtained withso-called luminescence bleach rate imaging (G. J. Brakenhoff, K.Visscher & G. Gijsbers, “Luminescence Bleach rate Imaging,” J. Microsc.,175 (1994), 154-161). in that imaging mode, the local rate ofphotobleaching rather than the luminescence intensity is used as acontrast parameter for image formation. Spatial non-uniformities of theexcitation intensity distribution lead to—apparent—variations of theobserved rate of photobleaching. With an experimentally determinedexcitation intensity distribution available, these apparent variationscan be corrected.

(iii) The method can be used for the correction of intensity variationsin an image of a specimen under investigation which are caused bynon-uniformities of the optical system of the microscope, a procedurereferred to as “shading correction.” In the simple case of aluminescently labelled specimen of which the detected luminescenceintensity is proportional to both the excitation intensity and thedetection efficiency, shading correction is accomplished by calculatingthe ratio of the image of the specimen under investigation and theproduct of the—relative or absolute—excitation intensity and detectionefficiency distributions. The fact that with the method, the excitationintensity and detection efficiency distributions are availableseparately implies that also in more complicated specimens, for examplespecimens in which non-linear dependencies occur, shading correction ispossible.

(iv) The method can be employed for the quantitative investigation ofspecimens. The intensity variations in a shading corrected image of aspecimen are independent of the microscope used to acquire the image andare determined only by specimen related factors such as theconcentrations of the luminophores in the specimen and their absorptionand emission characteristics. When shading correction is based on theabsolute excitation intensity and detection efficiency distributions,the intensities in the shading corrected image of a specimen can be usedto quantitatively determine these specimen related factors. For example,if a luminophore is available which can be used to luminescently label aspecimen and if the luminescence quantum yield and the absorption-crosssection of this luminophore are known and independent of themicro-environment within the specimen, the intensities in the shadingcorrected image can be used to quantitatively determine theconcentration of this luminophore in the specimen on a microscopiclevel.

(v) The excitation intensity and detection efficiency distributions canbe used for active image correction by modulating illumination anddetection parameters during image acquisition in such a way that aspatially uniform illumination and detection efficiency results. Thispossibility is important for bleach rate imaging, photoactivatableprocesses, assessment of biological cell damage, etcetera.

EXAMPLES

To demonstrate the applicability of the invention, a luminescent andphotobleachable calibration layer was prepared and used for shadingcorrection of images acquired with a confocal luminescence microscope.The luminescent and photobleachable calibration layer was based on theluminophore 4-dimethylamino-4′-nitrostilbene (DANS). Solutions of DANSand polymethylmethacrylate (PMMA) in chloroform were prepared and usedto spin-coat standard glass cover slips used for optical microscopy. Forthe investigation of the influence of the luminophore concentration onthe luminescence intensity and the rate of photobleaching, threecalibration layers were prepared with relative concentrations of 0.2,0.5 and 1.0. DANS in PMMA can be excited in the wavelength range <250nm-550 nm; it fluoresces in the wavelength range 500-850 nm. Uponirradiation, photobleaching of the fluorophore takes place, mainly dueto photo-oxidation.

The microscope used for this demonstration was an Olympus IMT-2 invertedmicroscope (Olympus Corporation, Lake Success, N.Y., USA), equipped withan INSIGHT PLUS bilateral confocal line scanning unit (MeridianInstruments Inc., Okemos, Mich., USA) and a 100×, NA=1.32, oil immersionobjective lens. Luminescence was excited at 488 nm, using an air cooledArgon ion laser (model 532, Omnichrome, Chino, Calif., USA). Theexcitation intensity could be varied by insertion of neutral densityfilters (NDFs) in the laser delivery path of the microscope. A total offour NDFs were available, with a transmittance ranging from 1% to 50%.The generated luminescence was detected with a cooled CCD camera (modelDDE/3200, Astromed, Cambridge, UK) through a long-pass filter with acut-off wavelength at 520 nm. A Hewlett-Packard model 725/50 workstation(Hewlett-Packard, Palo Alto, Calif., USA) was used for image collectionand exposure control via a mechanical laser shutter. Image analysis wascarried out on the same workstation, using the image processing packageScillmage (T. K. Ten Kate, R. van Balen, A. W. M. Smeulders, F. C. A.Groen & G. A. de Boer, “SCILIAM, a Multi-level Interactive ImageProcessing Environment,” Pattern Recognition Letters, 11 (1990),429-441).

For the investigation of the photobleaching characteristics of thecalibration layer, so-called “bleach curves” were determined byacquiring a series of images—a time series—from a certain part of thecalibration layer. The image exposure time was the same for all imagesin the time series, and no additional exposure occurred betweensuccessive images. From each time series, two quantities weredetermined: the mean initial luminescence intensity and the mean bleachrate. The mean initial luminescence intensity was calculated byaveraging the intensities in the first image of the time series. Themean bleach rate should ideally be determined by averaging the bleachrates calculated for each pixel in the first image of the time seriesfor a number of regions of interest (ROIs), which were chosenarbitrarily in the image. The data for each individual ROI were fittedwith a (mono-) exponential function. The mean bleach rate was obtainedas the mean of the individual ROI bleach rates.

For the luminescent layer it has been verified that its luminescence andphotobleaching characteristics conform to the requirements of themethod, i.e., the luminescence intensity in the first image of thecalibration layer—or the initial luminescence intensity—should beproportional to the excitation intensity, the luminophore concentration,and the image exposure time, its photobleaching should bemono-exponential, and its rate of photobleaching should be proportionalto the excitation intensity and independent of the luminophoreconcentration.

It was found that the conditions for the luminescence characteristicsare satisfied by the proposed calibration layer. The photobleaching ofthe calibration layer initially was not strictly mono-exponential;however, close to mono-exponential photobleaching of the calibrationlayer could be realised by “pre-bleaching” the layer. The data obtainedafter 180 sec of pre-bleaching were used to calculate “the rate ofphotobleaching,” which appeared to be proportional to the excitationintensity and independent of the luminophore concentration in the layer.Therefore, after suitable pre-bleaching, the requirements for thephotobleaching characteristics are fulfilled by the prepared calibrationlayer.

The excitation intensity distribution can be determined from two imagesof the calibration layer, which are separated by a time interval inwhich the calibration layer is partially bleached. To calculate therelative excitation intensity distribution, the first and second imageswere acquired with a pre-bleaching time of 180 sec and a bleach timeinterval of 150 sec, since in this time-interval the decrease of theluminescence intensity is described well with a mono-exponentialfunction. In these images, a stripe- and spot-like pattern can be seen,which is independent of the part of the calibration layer from which theimages were acquired. This indicates that the stripe- and spot-likepattern is caused by non-uniformities of the optical system of themicroscope. Inspection shows that the excitation intensity is notdistributed uniformly over the image region, but that a stripe-likepattern occurs. It was found that this pattern is caused by the dichroicmirror in the microscope. The relative magnitude of the variations inexcitation intensity over the image region can be expressed as thecoefficient of variation (CV)—the ratio of the standard deviation to themean. The measurement shows that in the relative—and absolute—excitationintensity distribution, variations of approximately 10% occur over theimage region. The CV is a measure of the variation across the entireimage. It should be pointed out that locally much larger variations canoccur.

The detection efficiency distribution can also be determined from twoimages—the first and second images—of the calibration layer. Therelative detection efficiency distribution is determined from theproduct distribution, which is proportional to the product of thedetection efficiency and luminophore concentration distributions. Theproduct distribution is determined from the first image of thecalibration layer and the already determined relative excitationintensity distribution. By calculating the product distribution fromdifferent, randomly chosen, parts of the calibration layer and averagingthe results, the relative detection efficiency distribution is obtained.By averaging a number of product distributions, measured at differentparts of the calibration layer, the relative detection efficiencydistribution is obtained. As already noted, this stripe-like pattern iscaused by the dichroic mirror in the microscope, and since this mirroris part of both the excitation and detection pathway, the same patternis visible in the relative excitation intensity and detection efficiencydistributions. Also visible are dark spots which cannot be seen in therelative excitation intensity distributions. These are probably causedby small dust particles or other irregularities in the detection pathwayof the microscope. The relative magnitude of the variations in detectionefficiency can be estimated by again taking the CV as a measure of thevariations. Variations of approximately 25% occurred in the relative—andabsolute—detection efficiency distributions. Again, locally thevariations can be much larger.

With the known relative excitation intensity and detection efficiencydistributions shading correction of an image of a specimen can becarried out by calculating the ratio of the image of the specimen to theproduct of the relative excitation intensity and detection efficiencydistributions.

Comparison before and after correction indicates a clear reduction ofthe intensity variations. Also visible is that a calibration layerfeature—the deliberately photobleached line-shaped region—is wellpreserved after the correction procedure, whereas the intensityvariations caused by the non-uniformities of the optical system havedisappeared. A different way is to visualize the effect of the shadingcorrection, in which case the intensities are plotted before and aftercorrection. It is clear from this that the intensity variations aftercorrection are significantly smaller than before correction. The effectof the shading correction was quantified by calculating the CV of theintensities in the images. The results show CVs of approximately 22% and4% before and after correction, respectively. This means that theshading correction procedure achieved a more than five-fold decrease ofthe intensity variations. Locally the decrease of the image intensityvariations obviously will be much larger.

What is claimed is:
 1. A calibration layer comprising an opticallytransparent polymer containing a photobleachable luminescent materialwherein the polymer contains less than 10 wt % of luminescent groupsoriginating from the luminescent material and has an optical attenuationof less than 0.3 absorption units in the wavelength region of 250 to1700 nm.
 2. The calibration layer of claim 1 wherein a photobleachableluminescent group contained in the photobleachable luminescent materialis covalently attached to a sidechain polymer the relative molar contentof which is lower than 10%.
 3. A method of calibration of an opticalimage device by: a. photobleaching the calibration layer of claim 1 as aseries of images from different parts of the calibration layer; b.calculating the mean initial luminescence intensity and the mean bleachrate for each series; c. calculating a detection efficacy distributionfrom the data of b; and d. using the decrease in luminescence as theresult of photobleaching between two subsequent images as a measure forcalibration.
 4. The method according to claim 3 wherein an optical orRaman microscope is calibrated.